toxins

Article The Effects of and Apamin on Airborne Fungi-Induced Chemical Mediator and Extracellular Matrix Production from Nasal Polyp Fibroblasts

Seung-Heon Shin 1,*, Mi-Kyung Ye 1, Sung-Yong Choi 1 and Kwan-Kyu Park 2

1 Department of Otolaryngology-Head and Neck Surgery, School of Medicine, Catholic University of Daegu, Daegu 42472, Korea; [email protected] (M.-K.Y.); [email protected] (S.-Y.C.) 2 Department of Pathology, School of Medicine, Catholic University of Daegu, Daegu 42472, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-53-650-4530; Fax: +82-53-650-4533

Academic Editor: Steve Peigneur Received: 20 September 2017; Accepted: 25 October 2017; Published: 27 October 2017

Abstract: Melittin and apamin are the main components of and they have been known to have anti-inflammatory and anti-fibrotic properties. The aim of this study was to evaluate the effect of melittin and apamin on airborne fungi-induced chemical mediator and extracellular matrix (ECM) production in nasal fibroblasts. Primary nasal fibroblasts were isolated from nasal polyps, which were collected during endoscopic sinus surgery. Nasal fibroblasts were treated with Alternaria and Aspergillus. The effects of melittin and apamin on the production of interleukin (IL)-6 and IL-8 were determined with enzyme linked immunosorbent assay. ECM mRNA and protein expressions were determined with the use of quantitative RT-PCR and Western blot. Alternaria-induced IL-6 and IL-8 production was significantly inhibited by apamin. However, melittin did not influence the production of IL-6 and IL-8 from nasal fibroblasts. Melittin or apamin significantly inhibited collagen type I, TIMP-1, and MMP-9 mRNA expression and protein production from nasal fibroblasts. Melittin and apamin inhibited Alternaria-induced phosphorylation of Smad 2/3 and p38 MAPK. Melittin and apamin can inhibit the fungi-induced production of chemical mediators and ECM from nasal fibroblasts. These results suggest the possible role of melittin and apamin in the treatment of fungi induced airway inflammatory diseases.

Keywords: melittin; apamin; Alternaria; Aspergillus; nasal fibroblast; chemical mediator; extracellular matrix

1. Introduction Nasal polyps are swellings and there is damage to the mucosal epithelium with inflammatory cell infiltration. Although the pathogenesis of nasal polyps is not fully understood, mucosal epithelial damage, extracellular matrix (ECM) accumulation, and increased local inflammatory mediators are the characteristic pathophysiologic findings of nasal polyps [1]. Fibroblasts are the main structural components of the nasal mucosa and they participate in the local immune response through the production of biological mediators which are involved in the recruitment of inflammatory cells and the cellular source of ECM proteins [2]. Fungi are ubiquitous saprophytes in nature and airway mucosa is exposed by inhalation of fungal spores. A fine immunologic balance to maintain a stable host–fungi relationship is important in order to maintain the physiologic condition, and disruption of the host immune response causes pathologic conditions of the airway. Fungi are commonly found in the nasal mucosa and relatively few species are implicated in airway inflammatory disease. Alternaria and Aspergillus are known common pathogens found in nasal secretion and they induce the production of chemical mediators from nasal epithelial

Toxins 2017, 9, 348; doi:10.3390/toxins9110348 www.mdpi.com/journal/toxins Toxins 2017, 9, 348 2 of 11

Toxins 2017, 9, 348 2 of 11 cells and fibroblasts [3,4]. TLR2, TLR4, and TLR5 seem to be important pattern recognition receptors forcommon fungi [pathogens3,5]. found in nasal secretion and they induce the production of chemical mediators fromBee nasal venom epithelial (BV) cells has and been fibroblasts used to[3,4]. treat TLR2, several TLR4,inflammatory and TLR5 seem diseases,to be important such pattern as rheumatoid arthritis,recognition tendonitis, receptors andfor fungi Parkinson’s [3,5]. disease [6,7]. BV is a complex mixture with peptides, enzymes, and biogenicBee venom amines (BV) has with been various used to pharmaceutical treat several inflammatory properties. diseases Melittin, such and as apamin rheumatoid are the main componentsarthritis, tendonitis, of BV peptide.and Parkinson’s Melittin disease comprises [6,7]. BV about is a complex 40–50% mixture of the driedwith peptides, BV and enzymes, has antibacterial, and biogenic amines with various pharmaceutical properties. Melittin and apamin are the main antiviral, anti-inflammatory, and anti-fibrotic properties [8–10]. Apamin also has anti-inflammatory components of BV peptide. Melittin comprises about 40–50% of the dried BV and has antibacterial, and anti-fibrotic properties in various cells [11]. antiviral, anti-inflammatory, and anti-fibrotic properties [8–10]. Apamin also has anti-inflammatory and Melittinanti-fibrotic and properties apamin in have various anti-fibrotic cells [11]. activities that suppress the pro-fibrotic gene and protein expressionMelittin through and apamin the have inhibition anti-fibrotic of TGF- activitiesβRII-Smad, that suppress ERK1/2, the pro-fibrotic and JNK gene phosphorylation and protein in rat kidneyexpression fibroblasts through [ 10the]. inhibition Melittin andof TGF- apaminβRII-Smad, have immunomodulatory ERK1/2, and JNK phosphorylation activities, and in in rat this study, wekidney evaluated fibroblasts the effect [10]. Melittin of melittin and and apamin apamin have on immunomodulatory airborne fungi induced activities, chemical and in mediatorthis study, and ECM productionwe evaluated in the nasal effect fibroblasts. of melittin and apamin on airborne fungi induced chemical mediator and ECM production in nasal fibroblasts. 2. Results 2. Results 2.1. The Cytotoxicity of BV, Melittin, and Apamin 2.1. The Cytotoxicity of BV, Melittin, and Apamin MTT assay was used to determine the cytotoxicity of these three agents. The cells were treated with MTT assay was used to determine the cytotoxicity of these three agents. The cells were treated various concentrations of BV (0.1 to 5 µg/mL), melittin (0.1 to 5 µg/mL), and apamin (0.1 to 10 µg/mL) with various concentrations of BV (0.1 to 5 μg/mL), melittin (0.1 to 5 μg/mL), and apamin (0.1 to 10 for 24 h. The viability of fibroblasts was significantly suppressed by BV at a concentration of 5 µg/mL μ/mL) for 24 h. The viability of fibroblasts was significantly suppressed by BV at a concentration of (55.85 μg/mL± 6.8%), (55.8 melittin ± 6.8%), at melittin a concentration at a concentration of 3 µg/mL of 3 (82.6 μg/mL± 8.3%), (82.6 ± and 8.3%), apamin and apamin at a concentration at a of 10concentrationµg/mL (55.8 of± 1011.7%) μg/mL (Figure(55.8 ± 111.7%)). Based (Figure on these 1). Based results, on these we used results, up towe 3 usedµg/mL up to of 3 BV,μg/mL 1 µ g/mL of melittin,of BV, 1 μ andg/mL 5 µofg/mL melittin, of apaminand 5 μg/mL for further of apamin experiments. for further experiments.

FigureFigure 1. EffectEffect of of bee bee venom, venom, melittin, melittin, and andapamin apamin on the on proliferation the proliferation of nasal of fbroblasts. nasal fbroblasts. Nasal Nasal fibroblasts were treated with various concentrations of various concentrations of (a) bee venom; (b) fibroblasts were treated with various concentrations of various concentrations of (a) bee venom; melittina, and (c) apamin for 24 h. Values are expressed as the mean ± SE of four independent (b) melittina; and (c) apamin for 24 h. Values are expressed as the mean ± SE of four independent experiments. 5 μg/mL of BV, 3 μg/mL of melittin, and 10 μg/mL of apamin inhibited the experiments. 5 µg/mL of BV, 3 µg/mL of melittin, and 10 µg/mL of apamin inhibited the proliferation proliferation of nasal fibroblasts. NC: negative control; BV: bee venom; M: melittin; A: apamin; μg: µ µ ofμg/mL; nasal * fibroblasts. p < 0.05. NC: negative control; BV: bee venom; M: melittin; A: apamin; g: g/mL; * p < 0.05.

2.2.2.2. TheThe EffectEffect of of BV, BV, Melittin, Melittin, and and Apamin Apamin on the on Production the Production of Chemical of Chemical Mediators Mediators WhenWhen thethe fibroblasts fibroblasts were were stimulated stimulated with withAlternaria Alternaria, IL-6, IL-6 (6476.1 (6476.1± 352.4 ± 352.4 pg/mL pg/mL at a concentrationat a ofconcentration 50 µg/mL, 3368.1of 50 μg/mL,± 247.5 3368.1 pg/mL ± 247.5 at apg/mL concentration at a concentration of 25 µg/mL) of 25 μ andg/mL) IL-8 and (7969.4 IL-8 (7969.4± 690.2 ± pg/mL 690.2 pg/mL at a concentration of 50 μg/mL, 2399.0 ± 175.5 pg/mL at a concentration of 25 μg/mL) at a concentration of 50 µg/mL, 2399.0 ± 175.5 pg/mL at a concentration of 25 µg/mL) production was production was significantly increased compared with that in the non-stimulated group (IL-6; significantly increased compared with that in the non-stimulated group (IL-6; 1812.1 ± 93.6 pg/mL, 1812.1 ± 93.6 pg/mL, IL-8; 1010.6 ± 132.4 pg/mL). However, Aspergillus did not significantly enhance IL-8;the production 1010.6 ± 132.4 of IL-6 pg/mL). and IL-8 However, from nasalAspergillus fibroblasts.did IL-6 not significantlyand IL-8 production enhance induced the production by of IL-6Alternaria and IL-8 was from significantly nasal fibroblasts. inhibited by IL-6 BV and and IL-8 apamin production in a dose induced dependent by Alternariamanner. However,was significantly inhibitedmelittin did by not BV influence and apamin the production in a dose of dependent IL-6 and IL-8 manner. from nasal However, fibroblasts melittin (Figure did 2). not influence the production of IL-6 and IL-8 from nasal fibroblasts (Figure2).

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FigureFigure 2. 2.Effect Effect ofof bee venom, melittin, melittin, and and apamin apamin on the on production the production of IL-6 of and IL-6 IL-8 and from IL-8 nasal from nasal fibroblasts.fibroblasts. NasalNasal fibroblastsfibroblasts were were treated treated with with AlternariaAlternaria and andAspergillusAspergillus for 24 forh with 24 hor with without or without variousvarious concentrations concentrations ofof thesethese threethree agents.agents. (a,d)) Alternaria enhancedenhanced IL-6 IL-6 an andd IL-8 IL-8 production production from nasalfrom fibroblasts nasal fibroblasts and the and production the production of IL-6 of andIL-6 and IL-8 IL-8 was was significantly significantly inhibited inhibited by by bee bee venom venom and (c,f) and (c,f) apamin; (b,e) Melittin did not influence the production of IL-6 and IL-8 from nasal apamin; (b,e) Melittin did not influence the production of IL-6 and IL-8 from nasal fibroblasts. Alt 50: fibroblasts. Alt 50: Alternaria 50 μg/mL; Asp 50: Aspergillus 50 μg/mL; NC: negative control; NT: Alternaria 50 µg/mL; Asp 50: Aspergillus 50 µg/mL; NC: negative control; NT: non-treated; BV: bee non-treated; BV: bee venom; Mel: melittin; Apa: apamin, μg: μg/mL; * p < 0.05 compared with venom; Mel: melittin; Apa: apamin, µg: µg/mL; * p < 0.05 compared with negative control; † p < 0.05 negative control; † p < 0.05 compared with the non-treated group. compared with the non-treated group. 2.3. The Effect of Melittin and Apamin on the Expression of ECM 2.3. The Effect of Melittin and Apamin on the Expression of ECM Collagen type I mRNA and protein expression was significantly increased with 50 μg/mL of Aspergillus.Collagen Aspergillus type I mRNA induced and collagen protein type expression I mRNA was and significantlyprotein expression increased was significantly with 50 µg/mL of Aspergillus.suppressed Aspergilluswhen treatedinduced with melittin. collagen Apamin type suppressed I mRNA both and Alternaria protein expressionand Aspergillus was induced significantly suppressedcollagen type when I mRNA treated and with protein melittin. expression Apamin (Figure suppressed 3). When both the Alternarianasal polypand fibroblastsAspergillus wereinduced stimulated with Alternaria TIMP-1 mRNA and protein expressions were significantly increased. collagen type I mRNA and protein expression (Figure3). When the nasal polyp fibroblasts were However, TIMP-1 mRNA and protein expressions were not significantly increased by stimulation stimulatedwith Aspergillus with .Alternaria TIMP-1 mRNATIMP-1 expression mRNA induced and protein by Alternaria expressions was significantly were significantly inhibited increased.by However,apamin TIMP-1in a dose mRNA dependent and protein manner, expressions and TIMP-1 were protein not significantly expression increasedwas also bysignificantly stimulation with Aspergillusinhibited. by TIMP-1 melittin mRNA and apamin expression in a dose induced dependent by Alternaria manner (Figurewas significantly 4). Alternaria inhibitedinduced MMP-9 by apamin in a dosemRNA dependent expression, manner, but Aspergillus and TIMP-1 did not protein induce expression MMP-9 mRNA was also expression significantly. Alternaria inhibited induced by melittin andMMP-9 apamin mRNA in a expression dose dependent was significantly manner (Figure inhibited4). byAlternaria meittin andinduced apamin. MMP-9 Although mRNA fungi expression,did butnotAspergillus influencedid the not production induce MMP-9 of MMP-9 mRNA protein, expression. melittinAlternaria and apamininduced tended MMP-9 to mRNAinhibit expressionthe wasproduction significantly of MMP-9 inhibited from bynasa meittinl polyp andfibroblasts apamin. (Figure Although 5). fungi did not influence the production of MMP-9 protein, melittin and apamin tended to inhibit the production of MMP-9 from nasal polyp fibroblasts (Figure5).

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Figure 3. EffectFigure of 3. melittinEffect of melittin and apaminand apamin on on the the expressionexpression of collagen of collagen type I mRNA type Ian mRNAd protein andin protein in nasal fibroblasts. Aspergillus induced collagen type I mRNA and protein expressions were nasal fibroblasts.significantlyAspergillus suppressedinduced by (a,b collagen) melittin and type (c,d I) mRNAapamin. Apamin and protein also significantly expressions suppressed were significantly suppressed bycollagen (a,b )type melittin I mRNA and expression (c,d) apamin. in negative Apamin control and also the Alternaria significantly stimulated suppressed group. Alt 50: collagen type I mRNA expressionAlternaria in 50 negative μg/mL; Asp control 50: Aspergillus and the 50Alternaria μg/mL; NC:stimulated negative control group.; NT: Alt non 50:-treatedAlternaria; Mel: 50 µg/mL; melittin; Apa: apamin; μg: μg/mL; *: p < 0.05 compared with negative control; † p < 0.05 compared Asp 50: Aspergilluswith the non50 µ-treatedg/mL; group. NC: negative control; NT: non-treated; Mel: melittin; Apa: apamin; µg: µg/mL; *: p < 0.05 compared with negative control; † p < 0.05 compared with the non-treated group. Toxins 2017, 9, x FOR PEER REVIEW 5 of 12

Figure 4. Effect of melittin and apamin on the expression of TIMP-1 mRNA and protein in nasal Figure 4. Effectfibroblasts. of melittin Alternaria andinduced apamin TIMP-1 onprotein the production expression was significantly of TIMP-1 inhibited mRNA by ( andb) melittin protein in nasal fibroblasts. Alternariaand (d) apamininduced; (c) Apamin TIMP-1 also protein significantly production suppressed was Alternaria significantly induced TIMP inhibited-1 mRNA by (b) melittin expression; (a) Melittin did no inhibit Alternaria induced TIMP-1 mRNA expression. Alt 50: and (d) apamin;Alternaria (c) Apamin 50 μg/mL also; Asp significantly 50: Aspergillus 50 suppressed μg/mL; NC:Alternaria negative controlinduced; NT: TIMP-1non-treated mRNA; Mel: expression; (a) Melittin didmelittin no; inhibitApa: apamin,Alternaria μg: μg/mLinduced; * p < 0.05 TIMP-1 compared mRNA with negative expression. control; † Alt p < 50:0.05 Alternariacompared 50 µg/mL; Asp 50: Aspergilluswith the non50 -µtreatedg/mL; group. NC: negative control; NT: non-treated; Mel: melittin; Apa: apamin, µg: µg/mL; * p < 0.05 compared with negative control; † p < 0.05 compared with the non-treated group.

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Figure 5. Effect of melittin and apamin on the expression of MMP-9 mRNA and protein in nasal Figure 5. Effect of melittin and apamin on the expression of MMP-9 mRNA and protein in nasal fibroblasts. Alternaria induced MMP-9 mRNA expression was significantly inhibited by (a) melittin fibroblasts.and (c) apamin.Alternaria (b,dinduced) Melittin MMP-9 and apamin mRNA tended expression to inhibit was the significantlyproduction of inhibitedMMP-9 protein. by (a) Alt melittin and50: (c) apamin.Alternaria (5b0, dμ)g Melittin/mL; Asp and 50: Aspergillus apamin tended 50 μg/mL to inhibit; NC: negative the production control; NT: of MMP-9 non-treated protein.; Mel Alt: 50: Alternariamelittin50; Aµpag/mL;: apamin; Asp μg: 50: μgAspergillus/mL; * p < 0.0550 comparedµg/mL; NC:with negative control control;; † p NT: < 0.05 non-treated; compared Mel: melittin;with the Apa: non apamin;-treated group.µg: µg/mL; * p < 0.05 compared with negative control; † p < 0.05 compared with the non-treated group. 2.4. Effect of Melittin and Apamin on Phosphorylation of Smad 2/3 and p38 MAPK 2.4. EffectTo of determine Melittin and the Apamininhibitory on mechanism Phosphorylation of ECM of Smadproduction, 2/3 and we p38 identified MAPK the effect of melittin and apamin on phosphorylation of Smad2/3 and p38 MAPK. Duration of treatment was 30 min for To determine the inhibitory mechanism of ECM production, we identified the effect of melittin and p-p38 and 60 min for pSmad 2/3. The densitometric quantification results showed that Alternaria apamin on phosphorylation of Smad2/3 and p38 MAPK. Duration of treatment was 30 min for p-p38 potently induced the activation of Smad 2/3 and p38 MAPK. When the fibroblasts were treated with and1 60 μ ming/mL for of pSmad melittin, 2/3. Smad The 2/3 densitometric (approximately quantification 30.9%), and results p38 MAPK showed (approximately that Alternaria 37.4%)potently inducedexpressions the activation were significantly of Smad 2/3 suppressed. and p38 MAPK. Apamin When also the significantly fibroblasts were suppressed treated Smad with 1 2/3µg/mL of melittin,(approximately Smad 2/3 27.4% (approximately and 36/4% at 1 30.9%), and 5 μ andg/mL p38 of apamin)MAPK (approximately and p38 MAPK (approxim 37.4%) expressionsately were29.2% significantly and 34.7% suppressed. at 1 and 5 μg Apamin/mL of apamin) also significantly expressions suppressed(Figure 6). Smad 2/3 (approximately 27.4% and 36/4% at 1 and 5 µg/mL of apamin) and p38 MAPK (approximately 29.2% and 34.7% at 1 and 5 µg/mL of apamin) expressions (Figure6).

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Figure 6. Effect of melittin and apamin on phosphorylation of Smad 2/3 and p38 MAPK expression Figure 6. Effect of melittin and apamin on phosphorylation of Smad 2/3 and p38 MAPK expression in in nasal fibroblasts. Phosphorylation of Smad 2/3 and p38 MAPK was measured using Western nasal fibroblasts. Phosphorylation of Smad 2/3 and p38 MAPK was measured using Western blotting blottingand density and analysis. density Alternariaanalysis. Alternariainduced Smad induced 2/3 andSmad p38 2/3 MAPK and p38 phosphorylation MAPK phosphorylation was significantly was significantlyinhibited by (ainhibited,c) melittin by and (a, (cb) ,dmelittin) apamin. and Alt ( 50:b,d)Alternaria apamin.50 Altµg/mL; 50: Alternaria Asp 50: Aspergillus50 μg/mL;50 Aspµg/mL; 50: AspergillusNC: negative 50 μ control;g/mL; NC: NT: non-treated;negative control; Mel: NT: melittin; non-treated; Apa: apamin; Mel: melittin;µg: µg/mL; Apa: apamin; * p < 0.05 μ comparedg: μg/mL; with* p < 0.05 negative compared control; with † p negative< 0.05 compared control; † with p < 0.05 the non-treatedcompared with group. the non-treated group.

3. Discussion 3. Discussion BV has been used as a traditional medicine with satisfactory results for the treatment of some BV has been used as a traditional medicine with satisfactory results for the treatment of inflammatory, cancer, and immune related diseases [6,7,12]. Melittin and apamin are the most some inflammatory, cancer, and immune related diseases [6,7,12]. Melittin and apamin are the well-known components of BV. Melittin is the main component of BV with hyaluronidase and most well-known components of BV. Melittin is the main component of BV with hyaluronidase [13]. Melittin is the active component of apitoxin with antimicrobial, and phospholipase A2 [13]. Melittin is the active component of apitoxin with antimicrobial, anti-inflammatory, and anti-atherosclerotic properties [8,9]. Apamin is a peptide with a anti-inflammatory, and anti-atherosclerotic properties [8,9]. Apamin is a peptide neurotoxin with crucial role in repetitive activities in neurons, inducing alpha-adrenergic, cholinergic, purinergic, a crucial role in repetitive activities in neurons, inducing alpha-adrenergic, cholinergic, purinergic, and neurotensin-induced relaxation [7,14]. In this study, we tried to determine the effect of melittin and neurotensin-induced relaxation [7,14]. In this study, we tried to determine the effect of melittin and and apamin on fungi induced chemical mediator and extraceullar matrix production from nasal apamin on fungi induced chemical mediator and extraceullar matrix production from nasal fibroblasts. fibroblasts. Fungi have been associated with upper and lower airway inflammatory diseases and Alternaria Fungi have been associated with upper and lower airway inflammatory diseases and Alternaria and Aspergillus are commonly found in the nasal secretion and the respiratory tract [15]. Fungi can and Aspergillus are commonly found in the nasal secretion and the respiratory tract [15]. Fungi can induce chemical mediator production through the interaction with toll-like receptors (TLRs) [3]. IL-6 induce chemical mediator production through the interaction with toll-like receptors (TLRs) [3]. IL-6 and IL-8 productions were increased with Alternaria and production of these chemical mediators and IL-8 productions were increased with Alternaria and production of these chemical mediators was significantly inhibited when the fibroblasts were treated with apamin. BV also significantly was significantly inhibited when the fibroblasts were treated with apamin. BV also significantly inhibited the production of IL-6 and IL-8. BV encompasses a mixture of many types of compounds, proteins, peptides, and enzymes. However, BV did not strongly inhibit chemical mediator

Toxins 2017, 9, 348 7 of 11 inhibited the production of IL-6 and IL-8. BV encompasses a mixture of many types of compounds, proteins, peptides, and enzymes. However, BV did not strongly inhibit chemical mediator production compared to apamin in nasal fibroblasts. IL-6 production was more strongly inhibited by BV (50.1% at 1 µg/mL of BV vs. 42.6% at 1 µg/mL of apamin), and IL-8 production was more strongly inhibited by apamin (28.8% at 1 µg/mL of BV vs. 38.7% at 1 µg/mL of apamin). Although we cannot explain the exact cause of this discrepancy, some components of BV may enhance the anti-inflammtory effect and the other components may suppress the anti-inflammatory effect of apamin. Anti-inflammatory properties of BV for treating skin disease, neurodegenerative disease, and joint diseases have been commonly studied, and these anti-inflammatory effects are associated with decreased expression of TLRs, chemical mediators, nitric oxides, and phosphorylation of inflammatory transcription factors [7]. According to the previous study, Alternaria induces the production of chemical mediators through TLR 2 and TLR5 [16]. Although we do not know the action mechanism of apamin and melittin, they may not influence the expression of TLR2 or 5 or the concentration of melittin used in this study may not enough to suppress the production of chemical mediators from nasal fibroblasts. The damage to mucosal epithelium, ECM accumulation, and inflammatory cell infiltration are important pathologic findings of nasal polyps [2]. Fibroblasts are the cellular source of ECM and they are involved in the development of nasal polyps. The MMP-9 level was elevated in nasal polyps and the TIMP-1 level was elevated in chronic rhinosinusitis [17]. We performed a kinetic study with Alternaria and Aspergillus for 8, 24, and 48 h. Collagen type I, TIMP-1, and MMP-9 mRNA expression levels were the highest at 24 h stimulation. Expression levels of these ECM proteins were highest at 6 h after stimulation with fungi. However, fungi did not influence the fibronectin mRNA and protein expression in nasal fibroblasts [18]. Therefore, we evaluated collagen type I, TIMP-1, and MMP-9 mRNA expression in nasal fibroblasts at 24 h and their protein expression at 6 h after stimulation with fungi. In this study, the expression pattern of ECM was different depending on the type of fungi used for stimulation. These differences may be associated with the unique molecular pattern, the peptides, or the immune triggering components of fungi [19]. Also, different fungi may interact with different pattern recognition receptors, such as TLRs, protease activated receptors, or G-protein-coupled receptors. TLR2, TLR4, and TLR9 are the main TLRs involved in sensing the fungal components [20]. Alternaria and Aspergillus enhance the production of chemical mediators through TLR4 in nasal epithelial cells [21]. Kao et al. suggested that TLR4 triggering activates the MAPK signaling pathway, which cross-talks with the Smad2 cascade and promotes the production of ECM [22]. When the nasal fibroblasts were stimulated with fungi, collagens type I, TIMP-1, and MMP-9 mRNA and/or protein expressions were increased. Also, this study showed that Alternaria can induce Smad 2/3 hyperphosphorylation in a TGF-β independent manner and p38 MAPK hyperphosphorylation. Our results suggest that Alternaria can directly activate the Smad 2/3 cascade or indirectly induce phosphorylation of Smad 2/3 through the TLR4/MAPK signaling pathway. Melittin and apamin have been known to inhibit ECM production and tissue fibrosis from and liver [10,11,23]. In this study, the inhibition of ECM expression in nasal fibroblasts by melittin and apamin differed depending on the types of ECM mRNA and protein. Although melittin and apamin show anti-fibrotic properties, they have different pharmacological characteristics, chemical structures, and they may control different signaling pathways. Melittin and apamin showed different patterns in suppressing the pro-fibrotic activity in TGF-β treated fibroblasts. Melittin attenuates fibrogenesis by inhibiting NF-κB and AP-1 dependent collagen type I and MMP-9 expression [10,24]. Apamin attenuates fibrogenesis by inhibiting phosphorylated Smad 2/3 and Smad dependent ECM deposition [25]. Melittin and apamin inhibited Alternaria induced phosphorylation of Smad 2/3 and MAPK in nasal fibroblasts. Although we do not know whether melittin and apamin directly suppress phosphorylation of Smad 2/3, melittin and apamin can directly or indirectly inhibit the Alternaria induced Smad 2/3 cascade. The principal finding of this study is the anti-inflammatory and anti-fibrotic effects of melittin and apamin. Fungi can induce production of chemical mediators and ECM deposition in nasal Toxins 2017, 9, 348 8 of 11

fibroblasts. The production of these chemical mediators and ECM production were inhibited by melittin and apamin. In particular, melittin and apamin inhibited ECM production through direct suppression of the Smad cascade or indirect inhibition of Smad 2/3 phosphorylation through the MAPK signaling pathway. These results suggest a novel pharmacological rationale for the treatment of fungi induced airway inflammatory diseases. Because BV contains melittin and apamin, BV may have strong anti-inflammatory and anti-fibrotic effects, and could be a good candidate as a therapeutic agent for airway inflammatory diseases.

4. Materials and Methods

4.1. Isolation of Primary Nasal Polyp Fibroblasts Primary nasal fibroblasts were isolated from 11 patients (7 men and 4 women; 43.5 ± 8.2 years) with chronic rhinosinusitis with nasal polyps during endoscopic sinus surgery. Subjects were excluded if they had an active inflammation, allergy, or aspirin hypersensitivity, had received antibiotics, , steroids, or other medications for at least four weeks preceding the surgery. Allergy status was defined using the skin prick test. The study was approved by the Institutional Review Board of Daegu Catholic University Medical Center. A duly completed written informed consent form that outlined the objectives of the research and experiments was obtained from each patient. The tissues were cut into 0.3 to 0.5 mm fragments and washed with phosphate buffered saline. These tissues were suspended and cultured in Dulbecco’s Modified Eagle’s Medium F-12 (DMEM/F-12) (Gibco, Grand Island, NY, USA) that contained 10% fetal bovine serum, penicillin at 100 U/mL, ◦ streptomycin at 100 µg/mL, and amphotericin B at 1.5 µg/mL at 37 C and 5% CO2. The second to third passages of fibroblasts were used for this experiment.

4.2. The Cytotoxic Effect of BV, Melittin, and Apamin on Nasal Polyp Fibroblasts The cytotoxic effect of BV (melittin comprise approximately 50% and apamin comprise 3% of dried BV) (Chung Jin Biotech Co., Ansan, Korea) [26], melittin (Enzo Life Sciences AG, Lausen, Switzerland), and apamin (Sigma-Aldrich, St. Louis, Mo, USA) was evaluated using a CellTiter-96® aqueous cell proliferation assay kit (Promega, Madison, WI, USA). On a 96-well microstate plate, NP fibroblasts were cultured in the presence of 0.1, 1, 3, and 5 µg/mL of BV, 0.1, 1, 3, and 5 µg/mL of melittin, and 0.1, ◦ 1, 5, and 10 µg/mL of apamin for 24 h at 37 C in a 5% CO2. The reduced tetrazolium compound produces a colored formazan product due to the mitochondrial activity in the cell. The amount of formazan is directly proportional to the number of viable cells. Color intensities were assessed with a fluorescence microplate reader at 490 nm.

4.3. The Effect of Bee Venom, Melittin, and Apamin on IL-6 and IL-8 Production from Nasal Polyp Fibroblasts The fibroblasts were incubated with endotoxin removed Alternaria alternate and Aspergillus fumigatus at 50 and 25 µg/mL, respectively (Greer Lab, Lenoir, NC, USA). After 24 h of stimulation, the cell culture supernatants and cells were harvested and stored at −70 ◦C until they were assayed. To determine the effect of melittin and apamin on the production of Interleukin (IL)-6 and IL-8, fibroblasts were incubated with or without various concentrations of melittin and apamin. IL-6 and IL-8 were quantified by using commercially available ELISA kits (R&D system, Minneapolis, MN, USA).

4.4. Real Time Reverse Transcription–Polymerase Chain Reaction (RT-PCR) for ECM mRNA from Nasal Polyp Fibroblasts Fibroblasts were exposed to fungi with or without melittin and apamin for 24 h. The total RNA was extracted from fibroblasts with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA, 1 µg, was reverse transcribed using SuPrimeScript RT Premix (Genetbio Inc., Daejeon, Korea) and Quantitative PCR was then carried out on a mini Toxins 2017, 9, 348 9 of 11 opticon system (Bio-Rad Lab., Hercules, CA, USA) according to the manufacturer’s protocol. The forward and reverse primers were as follows: β-actin, 5-ACAGGAAGTCCCTTGCCATC-3 and 5-AGGGAGACCAAAAGCCTTCA-3; α-SMA, 5-ATAGAACATGGCATCATCACCAAC-3, and 5-GGGCAACACGAAGCTCATTGTA-3; fibronectin, 5-GCCAGATGATGAGCTGCAC-3, and 5-GAGCAAATGGCACCGAGATA-3; tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) 5-CCTTATACCAGCGTTATGAGATCAA-3 and 5-AGTGATGTGCAAGAGTCCATCC-3; and matrix metalloproteinase-9 (MMP-9), 5-ATTTCTGCCAGGACCGCTTCTACT-3, and 5-CAGTTTGTATCCGGCAAACTGGCT-3. The cDNA was amplified with initial denaturation at 95 ◦C for 10 min, followed PCR for 40 cycles of 95 ◦C for 5 s, 58 ◦C for 30 s, and finally one cycle of melting curve following cooling at 60 ◦C for 60 s. To confirm the amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis. Analysis of relative gene expression was performed by evaluating q-RT-PCR data by the 2(-DDCt) method. The gene expression levels were determined by normalization relative to β-actin expression.

4.5. Western Blot Analysis of Nasal Polyp Fibroblasts Nasal fibroblast lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto NC membranes (Bio-Rad, Berkeley, CA, USA). Membranes were blocked with 5% skim milk solution and they were incubated with antibodies against MMP-9 (Cell signaling, Beverly, MA, USA), collagen type I, fibronectin, TIMP-1, phosphorylated Smad (pSmad) 2/3, p38 mitogen-activated protein kinase (MAPK), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation for 1 h, the membranes were washed and then treated with peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology). Bands were visualized using horseradish peroxidase conjugated secondary antibodies and an ECL system (Pierce, Rockford, IL, USA). The band densities were measured using the multi Gauge v.2.02 software (Fujifilm, Tokyo, Japan). The band intensities were expressed as a percentage of treated cells versus untreated cells.

4.6. Statistical Analysis The experimental data are presented as mean ± SE. The statistical significance of the differences between control and experimental data was analyzed using paired or unpaired Student’s t-test and one-way analysis of variance followed by Tukey’s test (SPSS ver. 21.0, SPSS Inc., Chicago, IL, USA). p value < 0.05 was considered to indicate a statistically significant difference. All results were obtained from at least four independent individuals and every experiment was performed in duplication.

Acknowledgments: This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01132501)” Rural Development Administration, Republic of Korea. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0023163). Author Contributions: Seung-Heon Shin conceived and designed the experiments; Mi-Kyung Ye and Sung-Yong Choi performed the experiments; Seung-Heon Shin and Kwan-Kyu Park analyzed the data; Mi-Kyung Ye contributed reagents/materials/analysis tools; Seung-Heon Shin and Sung-Yong Choi wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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